Project Summary Neuropeptides are a class of neurotransmitters or peptide hormones that generally activate G-Protein Coupled Receptors (GPCRs) and are involved in a large number of physiological and psychological processes in the brain and in the periphery. One such neuropeptide that we have identified computationally we called NPQ and was called Spexin by another group that independently identified the same molecule by a different computational method. Peptides derived from preproNPQ/Spexin are found in very localized regions of the brain, and we have determined that they are involved in pain response as well as having renal and cardiovascular actions. Very recently, it was discovered that NPQ/spexin is in the galanin peptide family and binds to and activates the Galanin Receptor 3 (GALR3). In fact, as we have validated, NPQ activates GALR3 at a far lower concentration than galanin activates this receptor. Based upon this discovery, we have hypothesized that NPQ, rather than galanin, is the primary neuropeptide that mediates the actions of GALR3, and through GALR3, NPQ has a role in acute and chronic pain and is a potential new target for as non- addicting analgesic. To test these hypotheses, Specific Aim 1 will use a series of in vitro assays to examine the interaction between NPQ peptides and GALR3 in vitro. GALR3 containing CHO cells that we have produced will be used to examine the binding and functional activity of NPQ and NPQ 53-70. Aim 1 will also use patch clamp electrophysiology to examine the ability of NPQ to block GABA release in slices from the ventrolateral PAG, a region with high levels of NPQ and GALR3, and known to be involved in pain perception. Specific Aim 2 will use in vivo assays to directly examine the involvement of NPQ and GALR3 in pain using in vivo models of acute thermal and inflammatory pain, and chronic neuropathic and inflammatory pain. Specific Aim 3 will screen combinatorial libraries for hit/lead generationto identify new small molecule agonists andbegin the process of lead identification for the development of novel therapeutics. Specific Aim 4 will further validate this target by examining the immunohistochemical localization of peptides derived from proNPQ, as well as the prohormone (proNPQ) itself. This will be compared with the localization of GALR3 and galanin. To determine how NPQ and GALR3 are involved in chronic neuropathic and inflammatory pain, NPQ peptides and GALR3 immunoreactivity, as well as mRNA levels, will compared in nave, spinal nerve ligated, and CFA treated mice. The involvement of GALR3 in the actions of NPQ will be verified by the use of GALR3 knockout mice. These experiments will verify the observation that NPQ activates GALR3, use in vitro and in vivo methods to examine how the NPQ/GALR3 system is involved in pain, and initiate a screening program to identify small molecule agonists as potential non-addicting analgesics through a novel GPCR target.